Evaluating Alfalfa Stress Tolerance

Project Title

Assessment of the Transcriptomic Response of Edited SPL8 Alfalfa Genotypes to Drought Stress.


Stacy Singer (AAFC Lethbridge) Stacy.singer@agr.gc.ca

Abdelali Hannoufa (AAFC London)

Status Project Code
Completed January, 2023 FRG.10.21


This team has successfully developed and used CRISPR/Cas gene editing technology targeting the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (SPL8) gene in alfalfa. These edited plants are more drought tolerant in the greenhouse. But many genes have more than one job, so shutting off SPL8 may affect more than just drought tolerance. In this case, they suspect the edited plants may have different levels of gibberellic acid, which is involved in leaf size and plant height; an effect that is likely distinct from drought tolerance. They want to see what else might be affected in the edited plants that could be responsible for the observed drought resilience.


  • Compare global transcript levels (gene expression) in control and edited alfalfa genotypes under both well-watered and drought conditions to see what genes are affected when SPL8 is targeted using CRISPR/Cas.

What you did

The team repeated the greenhouse drought experiment, harvested the appropriate tissues, extracted total RNA and assessed its quality, and sent the samples for RNA sequencing (3 biological replicates of each). The large amount of data obtained was analyzed using bioinformatics in order to identify pathways and genes of interest that differ between edited and control genotypes.

What you learned

From the large amount of data that was attained in this short 11 month study, it appears that drought resilience in these plants may be caused, at least in part, by changes in their response to oxidative stress, the expression of genes encoding particular transcription factors, and carbohydrate metabolism. In addition, it seems that different baseline levels of phytohormones, molecular antioxidants, and stress perception/signaling may also play a role.

What it means

While further research will be required to decipher the precise genes and metabolites responsible for drought tolerance in our plants, the knowledge and data gained from this study will provide an essential framework that will accelerate downstream breeding efforts to improve alfalfa’s drought tolerance in the future.